1. Field of the Invention
The present invention relates to a gene-entrapping liposome preparation and a process for the preparation thereof and more particularly, to the preparation which can be preserved over an extended period of time as well as the process for the preparation of same.
2. Related Arts
Liposomes have been expected as an actual preparation for gene therapy as quality control using liposomes is easier to manage than other gene therapy methods such as using a virus as a vector for introducing a gene, and the high degree of safety inherent in liposome methods. However, a plasmid DNA entrapped in liposomes shows low stability in a solution, and thus recent trends on study of liposomes have been occupied by a DNA/Lipids Complex method, wherein a solution containing gene recombinant plasmid DNA and a lipid solution for forming the liposomes are charged in a separate vial and then the plasmid DNA and lipids are mixed for preparing a required preparation, at the time of using the preparation.
The DNA/Lipids Complex method has disadvantages as it is difficult to prepare a uniform preparation, since the process for preparation is somewhat complicated. Therefore, there is great demand for a liposome preparation for gene therapy, which shows excellent stability in preservation, is easy in preparation at the time of using, and is constant in quality. It has been known that such multilamellar liposomes show higher gene entrapping efficiency, good expression of the gene and lower toxicity to cells and thus are useful as the vector for gene therapy in its effectiveness and safety. Their constitutive lipids are N-(xcex1-trimethyl ammonioacetyl)-didodecyl-D-glutamate chloride (TMAG), dilauroyl phosphatidylcholine (DLPC) and dioleoyl phosphatidyl ethanolamine (DOPE) and a molar ratio of the lipids is 1:2:2 [Japanese Patent 4-108391(A)]. However, the liposomes are not suitable for preservation over long period of time, since decomposition of DNA is apt to be caused, when the liposome preparation is preserved in a solution. An improvement per se in stability of DNA is possible by freezing the solution of liposomes entrapping the DNA, but there is a disadvantage that aggregation of the liposomes occurs during the freezing period of time, uniformity of the liposome in the defrosted solution at the time of using the same is spoiled, and thus biological activity of the liposome preparation becomes low.
Control of diameter of the liposomes is an important factor for carrying out gene therapies, since gene-entrapping liposomes having smaller diameter give toxicity to cells and larger liposomes show lower delivery efficiency of the entrapped gene to cells.
Therefore, an object of the invention is to provide a gene-entrapping liposome preparation which shows high gene catching efficiency, good expression of the gene, low toxicity to cells, high safety in use, excellence in stability during preservation, ease in conversion of form from preservation state to dosing state at the time of using, and substantially no reduction in biological activity during the conversion of form, as well as a process for the preparation of the gene-entrapping liposome preparation.
The inventors have intensively studied and investigated for overcoming the aforesaid problems. More particularly, the present invention is directed to liposomes obtained by adding an aqueous solution of a disaccharide to gene-entrapping liposomes and then freezing or lyophilized the solution containing the liposomes, to accomplish good restoration when the frozen or lyophilized liposomes are defrosted or suspended in water after preservation, and where a remarkable change in diameter cannot be recognized. Thus the liposomes do not cause aggregation thereof during the freezing and defrosting or lyophilizing and hydrating period and the gene-entrapping liposomes are excellent in expression of the gene DNA.
Therefore, the gene-entrapping liposome preparation which can be preserved over long period of time according to the invention is characterized by a frozen or lyophilized product containing the gene-entrapping liposomes and a disaccharide as a protector.
The process for the preparation of the gene-entrapping liposome preparation which can be preserved over long period of time according to the invention is characterized by adding an aqueous solution of disaccharide to the gene-entrapping liposomes and freezing or lyophilizing the same.
In the invention, it is preferable that constitutive lipids for the liposomes are TMAG, DLPC and DOPE and that molar ratio of the lipids is 1:2:2.
Such disaccharides can be listed as sucrose, trehalose, lactose, xylobiose, xytobiose, levanbiose, bicyanose, sambbiose, melibiose, epicerobiose, turanose, lactulose, rutinose, chondrosine and so on, but it is preferable to select at least one of sucrose and lactose. As an amount of the disaccharide, it is preferable in a range of 300-3333 g to 1 g of the gene which is entrapped in the liposomes, in both the cases of sucrose and lactose.
As mean diameter of the liposome, it is preferable in a range of 0.02-10 xcexcm, and more preferably in a range of 0.6-6 xcexcm.
The invention will be explained in more detail with reference to Test Examples. Followings are materials, process for preparing gene-entrapping liposomes and testing methods and so on used in the Test Examples.
Materials
(a) TMAG (manufactured by Sogo Pharmaceutical Co., Ltd.),
(b) DLPC (manufactured by Nichiyu Liposome Co., Ltd.),
(c) DOPE (manufactured by Nichiyu Liposome Co., Ltd.),
(d) Plasmid expressing human interferon xcex2 (pDRSV-INF xcex2) The plasmid was cloned by using E. coli DH5xcex1 and purified by using QIA Ultra 100 kit (manufactured by QIAGEN Company) to obtain the recombinant plasmid,
(e) Japanese Pharmacopoeia sucrose (manufactured by Kyowa Hakko Co., Ltd.),
(f) Lactose of superior quality (manufactured by Wako Pure Chemical Industries Ltd.),
(g) Japanese Pharmacopoeia lactose (manufactured by Iwaki Co., Ltd),
(h) Japanese Pharmacopoeia sorbitol (manufactured by Junsei Kagaku Co., Ltd.),
(i) Japanese Pharmacopoeia mannitol (manufactured by Kyowa Hakko Co., Ltd.),
(j) Xylitol of superior quality (manufactured by Wako Pure Chemical Industries Ltd.),
(k) Glucose of superior quality (manufactured by Wako Pure Chemical Industries Ltd.), and
(l) Japanese Pharmacopoeia magnesium gluconate (manufactured by Kyowa Hakko Co., Ltd.).
Process for the preparation of gene-entrapping liposomes
Lipids of TMAG, DLPC and DOPE were taken in molar ratio of 1:2:2 to dissolve in chloroform and then the solvent was removed through a condensation and drying in vacua. To this lipid mixture, pDRSV-IFN xcex2 and isotonic phosphate buffer (containing sodium chloride 13.7 mmol/l, sodium monohydrogenphosphate 8.7 mmol/l and sodium dihydrogenphosphate 1.4 mmol/l) were added to treat with polytron homogenizer at 15000 rpm and then subjecting to pressure filtration and centrifugal separation to prepare gene-entrapping liposomes. The liposomes contain DNA of about 1.4 mg/ml (measured by an absorption method) and lipid components of about 40 xcexc mol/ml (measured by HPLC), and have mean diameter of 1-2 xcexcm.
Freezing method
A suspension (1 ml portion) containing sucrose and gene-entrapping liposomes was prepared and dispensed into glass vials to freeze the suspension to obtain frozen samples.
Lyophilizing method
Prepared gene-entrapping liposomes (1 ml portion) were dispensed into glass vials and treated by a lyophilizer (manufactured by Kyowa Sinkuu Co., Ltd.) to obtain lyophilized samples.
Stability test
The frozen samples were preserved for 1-6 months at xe2x88x9220xc2x0 C., subjected to a periodical sampling, and the frozen samples were defrosted at room temperature to carry out tests on the following items:
(1) Measurement of diameter of the liposomes
Mean diameter of the liposomes was measured by using NICOMP M370 Submicronparticlesizer.
(2) Measurement of the amount of DNA remained
After solubilizing the liposomes with 10% Triton X-100, agarose-gel electrophoresis was carried out and ethidium bromide was added to measure fluorometrically an amount of DNA in the band of pDRSV-IFN xcex2. DNA purity is estimated by the ratio of fluorescent intensity in the band of pDRSV-IFN xcex2 to the total sum of fluorescent intensities in all bands obtained through the electrophoresis.
The lyophilized samples were preserved in a frozen or refrigerated state at xe2x88x9220, 5 and 10xc2x0 C., subjected to a periodical sampling, and tests were carried out on the following 4 items: Testing period of time is over 1-6 months or 4-12 months.
(1) Restoration
Distilled water of 1 ml was added to a lyophilized sample in the vial which was allowed to stand for 10 minutes and then shaken by hands to visually observe whether or not the suspension in the vial was uniform.
(2) Measurement of diameter of the liposomes
Mean diameter of the liposomes was measured by using NICOMP M370 Submicronparticlesizer.
(3) Measurement of the amount of DNA remained
After solubilizing the liposomes with 10% Triton X-100, agarose-gel electrophoresis was carried out and ethidium bromide was added to measure purity of DNA from the ratio of fluorescent intensity in the band of pDRSV-IFN xcex2 to the total sum of fluorescent intensities in all bands obtained through the electrophoresis. The ratio of DNA remaining was calculated on the basis of the purity of DNA before and after lyophilization.
(4) Expression test of human interferon xcex2
Liposomes diluted by D-MEM medium to a concentration of 15 ng/ml as DNA were added to U251SP strain, human malignant glicoma cells, and cultivated for 2 days at 37xc2x0 C. to measure an amount of expressed human interferon xcex2 by ELISA method.